Novel amylases and uses thereof

ABSTRACT

The invention relates to newly identified polynucleotide sequences comprising genes that encode novel amylases isolated from  Aspergillus niger . The invention features the full length nucleotide sequences of the novel genes, the cDNA sequences comprising the full length coding sequence of the novel amylases as well as the amino acid sequence of the full-length functional proteins and functional equivalents thereof. The invention also relates to methods of using these enzymes in industrial processes and methods of diagnosing fungal infections. Also included in the invention are cells transformed with a polynucleotide according to the invention and cells wherein an amylase according to the invention is genetically modified to enhance or reduce its activity and/or level of expression.

FIELD OF THE INVENTION

The invention relates to newly identified polynucleotide sequencescomprising genes that encode novel amylases isolated from Aspergillusniger. The invention features the full length nucleotide sequence of thenovel genes, the cDNA sequence comprising the full length codingsequences of the novel amylases as well as the amino acid sequences ofthe full-length functional proteins and functional equivalents thereof.The invention also relates to methods of using these enzymes inindustrial processes and methods of diagnosing fungal infections. Alsoincluded in the invention are cells transformed with a polynucleotideaccording to the invention and cells wherein an amylase according to theinvention is genetically modified to enhance its activity and/or levelof expression.

BACKGROUND OF THE INVENTION

Industrial processes for the hydrolysis of starch to glucose rely oninorganic acids or enzyme catalysis. The use of enzymes is preferredcurrently and offers a number of advantages associated with improvedyields and favourable economics. Enzymatic hydrolysis allows greatercontrol over amylolysis, the specifity of the reaction, and thestability of the generated products. The milder reaction conditionsinvolve lower temperatures and near-neutral pH, thus reducing unwantedside reactions. Fewer off-flavor and off-color compounds are produced,especially 5-hydroxy-2-methylfurfuraldehyde, anhydroglucose compounds,and undesirable salts. Enzymatic methods are favored because they alsolower energy requirements and eliminate neutralization steps.

Alpha-amylases (E.C. 3.2.1.1) or α-amylases catalyse the endohydrolysisof 1,4-alpha-glucosidic linkages in oligosaccharides andpolysaccharides. They are also known as 1,4-alpha-D-glucanglucanohydrolase, Taka-amylase, endoamylase or glycogenase. Alphaamylases act on starch, glycogen and related polysaccharides andoligosaccharides in a random manner; reducing groups are liberated inthe alpha-configuration.

Beta-amylases (E.C.3.2.1.2) catalyse the hydrolysis of1,4-alpha-glucosidic linkages in polysaccharides so as to removesuccessive maltose units from the non-reducing ends of the chains. Othernames are: 1,4-alpha-D-glucan maltohydrolase, Saccharogen amylase,Glycogenase. Beta amylases act on starch, glycogen and relatedpolysaccharides and oligosaccharides producing beta-maltose by aninversion.

Glucoamylases (E.C.3.2.1.3) catalyse the hydrolysis of terminal1,4-linked alpha-D-glucose residues successively from non-reducing endsof the chains with release of beta-D-glucose. Other names are: Glucan1,4-alpha-glucosidase. 1,4-alpha-D-glucan glucohydrolase.Amyloglucosidase. Gamma-amylase. Lysosomal alphaglucosidase.Exo-1,4-alpha-glucosidase. Most forms of the enzyme can rapidlyhydrolyse 1,6-alpha-D-glucosidic bonds when the next bond in sequence is1,4, and some preparations of this enzyme hydrolyse 1,6- and1,3-alpha-D-glucosidic bonds in other polysaccharides.

Amylases may conviently be produced in microorganisms. Microbialamylases are available from a variety of sources; Bacillus spec. are acommon source of bacterial enzymes, whereas fungal enzymes are commonlyproduced in Aspergillus spec.

The low pH optimum of most fungal amylases permits the convenient use ofacid conditions for the saccharification. Such conditions reduceunwanted isomerization reactions to fructose and other sugars that mayreduce the glucose yield. Moreover, acid conditions restrict the growthof contaminating microorganisms in the saccharification reactors.

Amylases may be used in a manifold of industrial applications, includingbaking, brewing, the production of corn syrup and alcohol as well as invinegar fermentation.

Malted wheat, barley, bacteria, and fungi are typical sources ofα-amylase for baking purposes. Fungal α-amylase is added to bread doughsin the form of diluted powders, prepacked doses, or water dispersibletablets. The enzyme may be added to flours at the bakery or, morerarely, at the mill itself. Malted wheat and barley also can serve assources of amylolytic activity when flours from these grains are blendedwith the final product at the mill. The properties of bacterialα-amylase permit its application to the production of coffee cake, fruitcake, brownies, cookies, snacks, and crackers. Fungal α-amylase, usuallyfrom A. oryzae, A. niger, A. awamori, or species of Rhizopus, is used tosupplement the amylolytic activity in flour. Enzymes from these sourcescan raise the levels of fermentable monosaccharides and disaccharides ofdough from a native level of 0.5% to concentrations that promote yeastgrowth. The sustained release of glucose and maltose by added fungal andendogenous enzymes provides the nutrients essential for yeast metabolismand gas production during panary fermentation. The A. oryzae α-amylaseis sometimes favored for baking applications over the bacterial enzymeobtained from Bacillus species since the fungal enzyme is heat labile at60-70° C. and does not survive the baking process. Its thermolabilityprevents enzymatic action on the gelatinised starch in the finished loafwhich would cause a soft or sticky crumb. Bacterial α-amylase is alsoused with good results, but its dose must be measured carefully to avoida bread with a gummy mouthfeel. Amylase supplementation is alsobeneficial and sometimes essential, since white bread flours contain6.7-10.5% damaged starch. The added enzyme degrades damaged, rupturedstarch granules that usually are present in bread flour more efficientlythan does wheat β-amylase (Bigelis R. in: Enzymes in Food processing,Nagodawithana and Reed Eds. Acad. Press Inc p121-158 and referencescited therein).

Amylase supplementation can improve other characteristics of breadquality, in addition to improving the quality of rolls, buns, andcrackers, when used during manufacturing processes for these bakedgoods. In bread baking, treatment with fungal or bacterial amylaselowers the viscosity of bread dough, thereby improving the ease ofmanipulation by manual workers or machines. Measured doses of enzymealso lower the compressibility of the loaf, producing a softer bread.Further, such processing increases the bread volume by reducing theviscosity of the gelling starch and allowing greater expansion duringbaking before protein denaturation and enzyme inactivation fix thevolume of the loaf. Favorable effects on taste, crust properties, andtoasting qualities are observed. The storage characteristics of breadsare changed also, yielding a product with a softer, more compressiblecrumb that firms more slowly and keeps longer, as determined by tastepanels. Amylolytic activity also may elevate the sugar concentration inbread and yield a preferred sweeter product with sensory advantages(Bigelis R. in: Enzymes in Food processing, Nagodawithana and Reed Eds.Acad. Press Inc p121-158 and references cited therein).

In brewing, added enzymes contribute to the action of endogenous barleyβ-amylase and aid in the starch digestion process. Such added enzymesare especially important when nonmalted cereal grains such as corn andrice, termed adjuncts, are used. Since these adjunct grains aredeficient in carbohydrases, fungal α-amylase and glucoamylase canincrease starch digestion, reduce the proportion of unmalted grain, andinsure a consistent quality of the mash. Amylase solubilizes barleyamylose and amylopectin, exposing these substrates to furtherdegradation by barley β-amylase. As a result, the levels of maltose andsmall dextrins are raised, eventually yielding the wort ingredients thatpromote yeast fermentation. Amylase preparations with lowtransglucosidase activity are favored since trace levels of this enzymegenerate isomaltose and panose, both of which are nonfermentable byyeast. The source of amylase activity for brewing applications isgenerally enzyme from Aspergillus species such as A. niger or A. oryzae.Protease from these sources may be added in concert with amylase tosolubilize protein and release amino acids essential for yeastproliferation. (Bigelis R. in: Enzymes in Food processing, Nagodawithanaand Reed Eds. Acad. Press Inc p121-158 and references cited therein)

In the above processes, it is advantageous to use enzymes that areobtained by recombinant DNA techniques. Such recombinant enzymes have anumber of advantages over their traditionally purified counterparts.Recombinant enzymes may be produced at a low cost price, high yield,free from contaminating agents like bacteria or viruses but also freefrom bacterial toxins or contaminating other enzyme activities.

Molecular cloning of amylases in fungi has been described. The DNA anddeduced amino acid sequences of certain alpha-amylases from Aspergillusoryzae, A. niger and A. shirousamil are given in Wirsel et al. Mol.Microbiol 1989, (1) 3-14, Boel et al., Biochemistry 1990 (29) 6244-6249,and Shibuya et al., Biosci. Biotech. Biochem. 1992 (56) 174179.Molecular cloning of an α-amylase from Bacillus amyloliquefaciens isdescribed by Takkinen et al. J. Biol. Chem. 1983, (258) 1007-1013.

It is important that amylases, in particular α-amylases, can be producedat low costs. This may be achieved by improving the productionefficiency (higher expression levels) or by providing enzymes with animproved specific activity (higher activity per mg of enzyme). It istherefore an object of the present invention to provide improved enzymeswith an improved production efficiency and/or improved specificactivity.

When α amylases are used as bread improvers, it is advantageous toprovide them, preferably together with other enzymes, in a liquidpreparation. Enzyme stabilisers like glycerol are a major cost factor ofliquid bread improvers and consequently there is a need for more stableα-amylases for use in such preparations in order to lower the amount ofstabilisers. It is also an object of the present invention to providemore stable α-amylases.

α-Amylases are often used in combination with ascorbic acid, which tendsto become unstable at higher pH values. α-Amylases on the other handbecome unstable at lower pH values. As a compromise between the tworequirements, such preparations are usually kept at a pH value around4.7. It would therefore be advantageous to have α-amylases with a lowerpH optimum and/or a higher stability at low pH values, preferably belowpH 4.7. The present invention provides such enzymes.

Ascorbic acid is used in combination with α-amylases in manyapplications where it is converted into a number of chelating agents,e.g. oxalate. Oxalate is able to bind Ca ions and since α-amylasesrequire Ca Ions for their stability, oxalate acts as a destabiliser forthese α-amylase enzyme preparations. It is therefore an objectunderlying the present invention to provide enzymes that are lessdependent on Ca ions for their stability.

Another characteristic of α-amylases according to the prior art is theirlimited thermostability. Fungal α-amylases are inactivated at about 65°C., therefore they are heat-inactivated at the beginning of the bakingprocess. Also, this property makes fungal α-amylases unsuited foractivity measurements in the Hagberg falling number method (AACC, 1983,Method 56-81 A) and the Brabender amylograph method (AACC, 1983, Method22-1). Also, prolonged storage at temperatures slightly above roomtemperature sometimes deteriorates enzyme activity. It is therefore anobject of the present invention to provide α-amylases with improvedthermostability.

OBJECT OF THE INVENTION

It is an object of the invention to provide novel polynucleotidesencoding improved novel amylases. A further object is to provideimproved naturally and recombinantly produced amylases as well asrecombinant strains producing these. Also fusion polypeptides are partof the invention as well as methods of making and using thepolynucleotides and polypeptides according to the invention.

SUMMARY OF THE INVENTION

The invention relates to isolated polypeptides having α-amylase activityand one or more characteristics selected from the group consisting of:

-   1) An isolated polypeptide having an amino acid sequence selected    from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID    NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO: 18 or functional    equivalents thereof,-   2) An isolated polypeptide obtainable by expressing a polynucleotide    having a nucleotide sequence selected from the group consisting of    SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5    or SEQ ID NO: 6 or from the group consisting of SEQ ID NO: 7, SEQ ID    NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12    or a vector comprising said polynucleotides or functional    equivalents thereof in an appropriate host cell, e.g. Aspergillus    niger.-   3) Polypeptide comprising a functional domain of a polypeptide    according to (1) or (2)-   4) An allelic variant of (1), (2) or (3),-   5) A fragment of (1), (2), (3) or (4)-   6) A polypeptide having improved specific activity and/or improved    production efficiency expressed as enzyme activity per mg of    purified enzyme or as enzyme activity per ml culture volume or per    mg of biomass produced-   7) A polypeptide with improved stability, preferably stable in the    presence of less than 50% glycerol, preferably less than 40%    glycerol, more preferably less than 30% glycerol, more preferably    less than 20% glycerol, more preferably less than 10% glycerol, most    preferably in the absence of glycerol-   8) A polypeptide stable at pH values below 4.7, preferably below pH    4.0, even more preferably below pH 3.5,-   9) A polypeptide with improved stability towards Ca ions.

It is expressly mentioned that α-amylases according to the invention mayhave one or more of the above characteristics. Methods for determiningspecific activity, production efficiency, stability, pH optimum and acidstability are well known in the art. Among others they may be found inthe materials and methods section of WO 00/60058.

The invention also relates to polynucleotides encoding any of thepolypeptides mentioned above.

More in particular, the invention provides for polynucleotides having anucleotide sequence that hybridises preferably under highly stringentconditions to a sequence having a nucleotide sequence selected from thegroup consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO: 5 or SEQ ID NO: 6 or from the group consisting of SEQ IDNO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQID NO: 12. Consequently, the invention provides nucleic acids that areabout 40%, preferably 65%, more preferably 70%, even more preferably75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homologous to any sequenceselected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ IDNO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 or from the groupconsisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10,SEQ ID NO: 11 or SEQ ID NO: 12.

In a more preferred embodiment the invention provides for such anisolated polynucleotide obtainable from a filamentous fungus, inparticular A. niger is preferred.

In one embodiment, the invention provides for an isolated polynucleotidecomprising a nucleic acid sequence encoding a polypeptide with having anamino acid sequence selected from the group consisting of SEQ ID NO: 13,SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO:18 or functional equivalents thereof.

In a further preferred embodiment, the invention provides an isolatedpolynucleotide encoding at least one functional domain of a polypeptidehaving an amino acid sequence selected from the group consisting of SEQID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 orSEQ ID NO: 18 or functional equivalents thereof.

In a preferred embodiment the invention provides an amylase gene havinga nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO:6. In another aspect the invention provides a polynucleotide, preferablya cDNA encoding an A. niger amylase having an amino acid sequenceselected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO: 18 or variants orfragments of that polypeptide. In a preferred embodiment the cDNA has anucleotide sequence selected from the group consisting of SEQ ID NO: 7,SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO:12 or functional equivalents thereof.

In an even further preferred embodiment, the invention provides for apolynucleotide comprising the coding sequence of the polynucleotidesaccording to the invention, preferred is a polynucleotide sequenceselected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ IDNO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 or from the groupconsisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10,SEQ ID NO: 11 or SEQ ID NO: 12.

The invention also relates to vectors comprising a polynucleotidesequence according to the invention and primers, probes and fragmentsthat may be used to amplify or detect the DNA according to theinvention.

In a further preferred embodiment, a vector is provided wherein thepolynucleotide sequence according to the invention is functionallylinked with regulatory sequences suitable for expression of the encodedamino acid sequence in a suitable host cell, such as A. niger or A.oryzea. The invention also provides methods for preparingpolynucleotides and vectors according to the invention.

The invention also relates to recombinantly produced host cells thatcontain heterologous or homologous polynucleotides according to theinvention.

In another embodiment, the invention provides recombinant host cellswherein the expression of an amylase according to the invention issignificantly increased or wherein the activity of the amylase isincreased.

In another embodiment the invention provides for a recombinantlyproduced host cell that contains heterologous or homologous DNAaccording to the invention and wherein the cell is capable of producinga functional amylase according to the invention, preferably a cellcapable of over-expressing the amylase according to the invention, forexample an Aspergillus strain comprising an increased copy number of agene or cDNA according to the invention.

In yet another aspect of the invention, a purified polypeptide isprovided. The polypeptides according to the invention include thepolypeptides encoded by the polynucleotides according to the invention.Especially preferred is a polypeptide having an amino acid sequenceselected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO: 18 or functionalequivalents thereof.

Fusion proteins comprising a polypeptide according to the invention arealso within the scope of the invention. The invention also providesmethods of making the polypeptides according to the invention.

The invention also relates to the use of the amylase according to theinvention in any industrial process as described herein

DETAILED DESCRIPTION OF THE INVENTION

Polynucleotides

The present invention provides polynucleotides encoding an alpha-amylasehaving an amino acid sequence selected from the group consisting of SEQID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 orSEQ ID NO: 18 or functional equivalents thereof. The sequence of thegenes encoding a protein according to the invention was determined bysequencing a genomic clone obtained from Aspergillus niger. Theinvention provides polynucleotide sequences comprising the gene encodingthe A niger alpha amylase as well as its complete cDNA sequence and itscoding sequence. Accordingly, the invention relates to an isolatedpolynucleotide comprising a nucleotide sequence selected from the groupconsisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,SEQ ID NO: 5 or SEQ ID NO: 6 or from the group consisting of SEQ ID NO:7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ IDNO: 12 or functional equivalents thereof.

More in particular, the invention relates to an isolated polynucleotidehybridisable under stringent conditions, preferably under highlystringent conditions, to a polynucleotide having a nucleotide sequenceselected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ IDNO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 or from the groupconsisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10,SEQ ID NO: 11 or SEQ ID NO: 12. Advantageously, such polynucleotides maybe obtained from filamentous fungi, in particular from Aspergillusniger. More specifically, the invention relates to an isolatedpolynucleotide having a nucleotide sequence having a nucleotide sequenceselected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ IDNO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 or from the groupconsisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10,SEQ ID NO: 11 or SEQ ID NO: 12.

The invention also relates to an isolated polynucleotide encoding atleast one functional domain of a polypeptide having an amino acidsequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO: 18 orfunctional equivalents thereof.

As used herein, the terms “gene” and “recombinant gene” refer to nucleicacid molecules which may be isolated from chromosomal DNA, which includean open reading frame encoding a protein, e.g. an A. niger amylase. Agene may include coding sequences, non-coding sequences, introns andregulatory sequences. Moreover, a gene refers to an isolated nucleicacid molecule as defined herein.

A nucleic acid molecule of the present invention, such as a nucleic acidmolecule having a nucleotide sequence selected from the group consistingof SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5or SEQ ID NO: 6 or from the group consisting of SEQ ID NO: 7, SEQ ID NO:8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12 or afunctional equivalent thereof, can be isolated using standard molecularbiology techniques and the sequence information provided herein. Forexample, using all or portion of the nucleic acid sequence of SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6,SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11or SEQ ID NO: 12 as a hybridization probe, nucleic acid moleculesaccording to the invention can be isolated using standard hybridizationand cloning techniques (e.g., as described in Sambrook, J., Fritsh, E.F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed.,Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989).

Moreover, a nucleic acid molecule encompassing all or a portion of SEQID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ IDNO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ IDNO: 11 or SEQ ID NO: 12 can be isolated by the polymerase chain reaction(PCR) using synthetic oligonucleotide primers designed based upon thesequence information provided herein.

A nucleic acid of the invention can be amplified using cDNA, mRNA oralternatively, genomic DNA, as a template and appropriateoligonucleotide primers according to standard PCR amplificationtechniques. The nucleic acid so amplified can be cloned into anappropriate vector and characterized by DNA sequence analysis.

Furthermore, oligonucleotides corresponding to or hybridisable tonucleotide sequences according to the invention can be prepared bystandard synthetic techniques, e.g., using an automated DNA synthesizer.

In a preferred embodiment, an isolated nucleic acid molecule of theinvention comprises the nucleotide sequence shown in SEQ ID NO: 7, SEQID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12.The sequence information provided in SEQ ID NO: 7, SEQ ID NO: 8, SEQ IDNO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12 corresponds to thecoding region of the A. niger alpha amylases genes provided in SEQ IDNO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ IDNO: 6 respectively. This cDNA comprises sequences encoding the A. nigeralpha amylases having an amino acid sequence selected from the groupconsisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:16, SEQ ID NO: 17 or SEQ ID NO: 18 respectively.

In another preferred embodiment, an isolated nucleic acid molecule ofthe invention comprises a nucleic acid molecule which is a complement ofthe nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8,SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12 or afunctional equivalent of these nucleotide sequences.

A nucleic acid molecule which is complementary to another nucleotidesequence is one which is sufficiently complementary to the othernucleotide sequence such that it can hybridize to the other nucleotidesequence thereby forming a stable duplex.

One aspect of the invention pertains to isolated nucleic acid moleculesthat encode a polypeptide of the invention or a functional equivalentthereof such as a biologically active fragment or domain, as well asnucleic acid molecules sufficient for use as hybridisation probes toidentify nucleic acid molecules encoding a polypeptide of the inventionand fragments of such nucleic acid molecules suitable for use as PCRprimers for the amplification or mutation of nucleic acid molecules.

An “isolated polynucleotide” or “isolated nucleic acid” is a DNA or RNAthat is not immediately contiguous with both of the coding sequenceswith which it is immediately contiguous (one on the 5′ end and one onthe 3′ end) in the naturally occurring genome of the organism from whichit is derived. Thus, in one embodiment, an isolated nucleic acidincludes some or all of the 5′ non-coding (e.g., promotor) sequencesthat are immediately contiguous to the coding sequence. The termtherefore includes, for example, a recombinant DNA that is incorporatedinto a vector, into an autonomously replicating plasmid or virus, orinto the genomic DNA of a prokaryote or eukaryote, or which exists as aseparate molecule (e.g., a cDNA or a genomic DNA fragment produced byPCR or restriction endonuclease treatment) independent of othersequences. It also includes a recombinant DNA that is part of a hybridgene encoding an additional polypeptide that is substantially free ofcellular material, viral material, or culture medium (when produced byrecombinant DNA techniques), or chemical precursors or other chemicals(when chemically synthesized). Moreover, an “isolated nucleic acidfragment” is a nucleic acid fragment that is not naturally occurring asa fragment and would not be found in the natural state.

As used herein, the terms “polynucleotide” or “nucleic acid molecule”are intended to include DNA molecules (e.g., cDNA or genomic DNA) andRNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated usingnucleotide analogs. The nucleic acid molecule can be single-stranded ordouble-stranded, but preferably is double-stranded DNA. The nucleic acidmay be synthesized using oligonucleotide analogs or derivatives (e.g.,inosine or phosphorothioate nucleotides). Such oligonucleotides can beused, for example, to prepare nucleic acids that have alteredbase-pairing abilities or increased resistance to nucleases.

Another embodiment of the invention provides an isolated nucleic acidmolecule which is antisense to a nucleic acid molecule according to theinvention. Also included within the scope of the invention are thecomplement strands of the nucleic acid molecules described herein.

In certain applications it may be advantageous to have products that arefree of amylase activity. Such products may be produced inmicroorganisms wherein an amylase gene according to the invention iseliminated or wherein its activity is reduced. Such microorganisms maybe obtained by recombinant DNA technology, for instance by knocking outthe expression of a gene according to the invention. Amylase deficientmutants may be advantageously used for the production of milk clottingenzymes where contamination with amylases is undesired.

Instead of elimination of amylase activities via disruption ormutagenesis, reduced amylase activity can also be achieved viadown-regulation of the amylase activities. This may be achieved bygenetically altering the promoter or other regulatory sequences of thegene(s) according to the invention. With the help of the sequenceinformation provided herein, the skilled person will know how to achievethe goal of providing mutant microorganisms with reduced or eliminatedamylase activity.

Sequencing Errors

The sequence information as provided herein should not be so narrowlyconstrued as to require inclusion of erroneously identified bases. Thespecific sequences disclosed herein can be readily used to isolate thecomplete gene from filamentous fungi, in particular A niger which inturn can easily be subjected to further sequence analyses therebyidentifying sequencing errors.

Unless otherwise indicated, all nucleotide sequences determined bysequencing a DNA molecule herein were determined using an automated DNAsequencer and all amino acid sequences of polypeptides encoded by DNAmolecules determined herein were predicted by translation of a DNAsequence determined as above. Therefore, as is known in the art for anyDNA sequence determined by this automated approach, any nucleotidesequence determined herein may contain some errors. Nucleotide sequencesdetermined by automation are typically at least about 90% identical,more typically at least about 95% to at least about 99.9% identical tothe actual nucleotide sequence of the sequenced DNA molecule. The actualsequence can be more precisely determined by other approaches includingmanual DNA sequencing methods well known in the art. As is also known inthe art, a single insertion or deletion in a determined nucleotidesequence compared to the actual sequence will cause a frame shift intranslation of the nucleotide sequence such that the predicted aminoacid sequence encoded by a determined nucleotide sequence will becompletely different from the amino acid sequence actually encoded bythe sequenced DNA molecule, beginning at the point of such an insertionor deletion.

The person skilled in the art is capable of identifying such erroneouslyidentified bases and knows how to correct for such errors.

Nucleic Acid Fragments, Probes and Primers

A nucleic acid molecule according to the invention may comprise only aportion or a fragment of the nucleic acid sequence shown in SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6,SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11or SEQ ID NO: 12, for example a fragment which can be used as a probe orprimer or a fragment encoding a portion of a protein according to theinvention. The nucleotide sequence determined from the cloning of thealpha amylase gene and cDNA allows for the generation of probes andprimers designed for use in identifying and/or cloning other alphaamylase family members, as well as homologues from other species. Theprobe/primer typically comprises substantially purified oligonucleotidewhich typically comprises a region of nucleotide sequence thathybridizes preferably under highly stringent conditions to at leastabout 12 or 15, preferably about 18 or 20, preferably about 22 or 25,more preferably about 30, 35, 40, 45, 50, 55, 60, 65, or 75 or moreconsecutive nucleotides of a nucleotide sequence shown in SEQ ID NO: 1,SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6,SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11or SEQ ID NO: 12 or of a functional equivalent thereof.

Probes based on the nucleotide sequences provided herein can be used todetect transcripts or genomic sequences encoding the same or homologousproteins for instance in other organisms. In preferred embodiments, theprobe further comprises a label group attached thereto, e.g., the labelgroup can be a radioisotope, a fluorescent compound, an enzyme, or anenzyme cofactor. Such probes can also be used as part of a diagnostictest kit for identifying cells which express an alpha-amylase.

Identity & Homology

The terms “homology” or “percent identity” are used interchangeablyherein. For the purpose of this invention, it is defined here that inorder to determine the percent identity of two amino acid sequences orof two nucleic acid sequences, the sequences are aligned for optimalcomparison purposes (e.g., gaps can be introduced in the sequence of afirst amino acid or nucleic acid sequence for optimal alignment with asecond amino acid sequence or nucleic add sequence). The amino acidresidues or nucleotides at corresponding amino acid positions ornucleotide positions are then compared. When a position in the firstsequence is occupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences (i.e., % identity=number of identical positions/totalnumber of positions (i.e. overlapping positions)×100). Preferably, thetwo sequences are the same length.

The skilled person will be aware of the fact that several differentcomputer programms are available to determine the homology between twosequences. For instance, a comparison of sequences and determination ofpercent identity between two sequences can be accomplished using amathematical algorithm. In a preferred embodiment, the percent identitybetween two amino acid sequences is determined using the Needleman andWunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package (formerlyavailable at http://www.gcg.com now at http://www.accelrys.com), usingeither a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16,14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. Theskilled person will appreciate that all these different parameters willyield slightly different results but that the overall percentageidentity of two sequences is not significantly altered when usingdifferent algorithms.

In yet another embodiment, the percent identity between two nucleotidesequences is determined using the GAP program in the GCG softwarepackage (formerly available at http://www.gcg.com now athttp://www.accelrys.com), using a NWSgapdna.CMP matrix and a gap weightof 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. Inanother embodiment, the percent identity two amino acid or nucleotidesequence is determined using the algorithm of E. Meyers and W. Miller(CABIOS, 4:11-17 (1989) which has been incorporated into the ALIGNprogram (version 2.0) (available at:http://vega.igh.cnrs.fr/bin/align-guess.cgi) using a PAM120 weightresidue table, a gap length penalty of 12 and a gap penalty of 4.

The nucleic acid and protein sequences of the present invention canfurther be used as a “query sequence” to perform a search against publicdatabases to, for example, identify other family members or relatedsequences. Such searches can be performed using the NBLAST and XBLASTprograms (version 2.0) of Altschul, et al. (1990) J. Mol. Biol.215:403-10. BLAST nucleotide searches can be performed with the NBLASTprogram, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to nucleic acid molecules of the invention. BLAST proteinsearches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to proteinmolecules of the invention. To obtain gapped alignments for comparisonpurposes, Gapped BLAST can be utilized as described in Altschul et al.,(1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST andGapped BLAST programs, the default parameters of the respective programs(e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.

Hybridisation

As used herein, the term “hybridizing” is intended to describeconditions for hybridization and washing under which nucleotidesequences at least about 50%, at least about 40%, at least about 70%,more preferably at least about 80%, even more preferably at least about85% to 90%, more preferably at least 95% homologous to each othertypically remain hybridized to each other.

A preferred, non-limiting example of such hybridization conditions arehybridization in 6× sodium chloride/sodium citrate (SSC) at about 45°C., followed by one or more washes in 1×SSC, 0.1% SDS at 50° C.,preferably at 55° C., preferably at 60° C. and even more preferably at65° C.

Highly stringent conditions include, for example, hybridizing at 68° C.in 5×SSC/5× Denhardt's solution/1.0% SDS and washing in 0.2×SSC/0.1% SDSat room temperature. Alternatively, washing may be performed at 42° C.

The skilled artisan will know which conditions to apply for stringentand highly stringent hybridisation conditions. Additional guidanceregarding such conditions is readily available in the art, for example,in Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, ColdSpring Harbor Press, N.Y.; and Ausubel et al. (eds.), 1995, CurrentProtocols in Molecular Biology, (John Wiley & Sons, N.Y.).

Of course, a polynucleotide which hybridizes only to a poly A sequence(such as the 3′ terminal poly(A) tract of mRNAs), or to a complementarystretch of T (or U) resides, would not be included in a polynucleotideof the invention used to specifically hybridize to a portion of anucleic acid of the invention, since such a polynucleotide wouldhybridize to any nucleic acid molecule containing a poly (A) stretch orthe complement thereof (e.g., practically any double-standed cDNAclone).

Obtaining Full Length DNA from Other Organisms

In a typical approach, cDNA libraries constructed from other organisms,e.g. filamentous fungi, in particular from the species Aspergillus canbe screened.

For example, Aspergillus strains can be screened for homologouspolynucleotides by Northern blot analysis. Upon detection of transcriptshomologous to polynucleotides according to the invention, cDNA librariescan be constructed from RNA isolated from the appropriate strain,utilizing standard techniques well known to those of skill in the art.Alternatively, a total genomic DNA library can be screened using a probehybridisable to a polynucleotide according to the invention.

Homologous gene sequences can be isolated, for example, by performingPCR using two degenerate oligonucleotide primer pools designed on thebasis of nucleotide sequences as taught herein.

The template for the reaction can be cDNA obtained by reversetranscription of mRNA prepared from strains known or suspected toexpress a polynucleotide according to the invention. The PCR product canbe subcloned and sequenced to ensure that the amplified sequencesrepresent the sequences of a new alpha amylase nucleic acid sequence, ora functional equivalent thereof.

The PCR fragment can then be used to isolate a full length cDNA clone bya variety of known methods. For example, the amplified fragment can belabeled and used to screen a bacteriophage or cosmid cDNA library.Alternatively, the labeled fragment can be used to screen a genomiclibrary.

PCR technology also can be used to isolate full length cDNA sequencesfrom other organisms. For example, RNA can be isolated, followingstandard procedures, from an appropriate cellular or tissue source. Areverse transcription reaction can be performed on the RNA using anoligonucleotide primer specific for the most 5′ end of the amplifiedfragment for the priming of first strand synthesis.

The resulting RNA/DNA hybrid can then be “tailed” (e.g., with guanines)using a standard terminal transferase reaction, the hybrid can bedigested with RNase H, and second strand synthesis can then be primed(e.g., with a poly-C primer). Thus, cDNA sequences upstream of theamplified fragment can easily be isolated. For a review of usefulcloning strategies, see e.g., Sambrook et al., supra; and Ausubel etal., supra.

Vectors

Another aspect of the invention pertains to vectors, preferablyexpression vectors, containing a nucleic acid encoding a proteinaccording to the invention or a functional equivalent thereof. As usedherein, the term “vector” refers to a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked. One typeof vector is a “plasmid”, which refers to a circular double stranded DNAloop into which additional DNA segments can be ligated. Another type ofvector is a viral vector, wherein additional DNA segments can be ligatedinto the viral genome. Certain vectors are capable of autonomousreplication in a host cell into which they are introduced (e.g.,bacterial vectors having a bacterial origin of replication and episomalmammalian vectors). Other vectors (e.g., non-episomal mammalian vectors)are integrated into the genome of a host cell upon introduction into thehost cell, and thereby are replicated along with the host genome.Moreover, certain vectors are capable of directing the expression ofgenes to which they are operatively linked. Such vectors are referred toherein as “expression vectors”. In general, expression vectors ofutility in recombinant DNA techniques are often in the form of plasmids.The terms “plasmid” and “vector” can be used interchangeably herein asthe plasmid is the most commonly used form of vector. However, theinvention is intended to include such other forms of expression vectors,such as viral vectors (e.g., replication defective retroviruses,adenoviruses and adeno-associated viruses), which serve equivalentfunctions.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell, which means that the recombinant expression vectorincludes one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, which is operatively linked to thenucleic acid sequence to be expressed. Within a recombinant expressionvector, “operatively linked” is intended to mean that the nucleotidesequence of interest is linked to the regulatory sequence(s) in a mannerwhich allows for expression of the nucleotide sequence (e.g., in an invitro transcription/translation system or in a host cell when the vectoris introduced into the host cell). The term “regulatory sequence” isintended to include promoters, enhancers and other expression controlelements (e.g., polyadenylation signal). Such regulatory sequences aredescribed, for example, in Goeddel; Gene Expression Technology: Methodsin Enzymology 185, Academic Press, San Diego, Calif. (1990). Regulatorysequences include those which direct constitutive expression of anucleotide sequence in many types of host cells and those which directexpression of the nucleotide, sequence only in a certain host cell (e.g.tissue-specific regulatory sequences). It will be appreciated by thoseskilled in the art that the design of the expression vector can dependon such factors as the choice of the host cell to be transformed, thelevel of expression of protein desired, etc. The expression vectors ofthe invention can be introduced into host cells to thereby produceproteins or peptides, encoded by nucleic acids as described herein (e.g.alpha-amylases, mutant alpha amylases, fragments thereof, variants orfunctional equivalents thereof, fusion proteins, etc.).

The recombinant expression vectors of the invention can be designed forexpression of alpha amylases in prokaryotic or eukaryotic cells. Forexample, a protein according to the invention can be expressed inbacterial cells such as E. coli, insect cells (using baculovirusexpression vectors) yeast cells or mammalian cells. Suitable host cellsare discussed further in Goeddel, Gene Expression Technology: Methods inEnzymology 185, Academic Press, San Diego, Calif. (1990). Alternatively,the recombinant expression vector can be transcribed and translated invitro, for example using T7 promoter regulatory sequences and T7polymerase.

Expression vectors useful in the present invention include chromosomal-,episomal- and virus-derived vectors e.g., vectors derived from bacterialplasmids, bacteriophage, yeast episome, yeast chromosomal elements,viruses such as baculoviruses, papova viruses, vaccinia viruses,adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses,and vectors derived from combinations thereof, such as those derivedfrom plasmid and bacteriophage genetic elements, such as cosmids andphagemids.

The DNA insert should be operatively linked to an appropriate promoter,such as the phage lambda PL promoter, the E. coli lac, trp and tacpromoters, the SV40 early and late promoters and promoters of retroviralLTRs, to name a few. Other suitable promoters will be known to theskilled person. In a specific embodiment, promoters are preferred thatare capable of directing a high expression level of amylases infilamentous fungi. Such promoters are known in the art. The expressionconstructs may contain sites for transcription initiation, termination,and, in the transcribed region, a ribosome binding site for translation.The coding portion of the mature transcripts expressed by the constructswill include a translation initiating AUG at the beginning and atermination codon appropriately positioned at the end of the polypeptideto be translated.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid (e.g., DNA) into a host cell, including calcium phosphate orcalcium chloride co-percipitation, DEAE-dextran-mediated transfection,transduction, infection, lipofection, cationic lipidmediatedtransfection or electroporation. Suitable methods for transforming ortransfecting host cells can be found in Sambrook, et al. (MolecularCloning: A Laboratory Manual, 2^(nd) , ed. Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989), Davis et al., Basic Methods in Molecular Biology (1986) andother laboratory manuals.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest.Preferred selectable markers include those which confer resistance todrugs, such as G418, hygromycin and methatrexate. Nucleic acid encodinga selectable marker can be introduced into a host cell on the samevector as that encoding a protein according to the invention or can beintroduced on a separate vector. Cells stably transfected with theintroduced nucleic acid can be identified by drug selection (e.g. cellsthat have incorporated the selectable marker gene will survive, whilethe other cells die).

Expression of proteins in prokaryotes is often carried out in E. coliwith vectors containing constitutive or inducible promoters directingthe expression of either fusion or non-fusion proteins. Fusion vectorsadd a number of amino acids to a protein encoded therein, e.g. to theamino terminus of the recombinant protein. Such fusion vectors typicallyserve three purposes: 1) to increase expression of recombinant protein;2) to increase the solubility of the recombinant protein; and 3) to aidin the purification of the recombinant protein by acting as a ligand inaffinity purification. Often, in fusion expression vectors, aproteolytic cleavage site is introduced at the junction of the fusionmoiety and the recombinant protein to enable separation of therecombinant protein from the fusion moiety subsequent to purification ofthe fusion protein. Such enzymes, and their cognate recognationsequences, include Factor Xa, thrombin and enterokinase.

As indicated, the expression vectors will preferably contain selectablemarkers. Such markers include dihydrofolate reductase or neomycinresistance for eukarotic cell culture and tetracyline or ampicillingresistance for culturing in E. coli and other bacteria. Representativeexamples of appropriate host include bacterial cells, such as E. coli,Streptomyces and Salmonella typhimurium; fungal cells, such as yeast;insect cells such as Drosophila S2 and Spodoptera Sf9; animal cells suchas CHO, COS and Bowes melanoma; and plant cells. Appropriate culturemedia and conditions for the above-described host cells are known in theart.

Among vectors preferred for use in bacteria are pQE70, pQE60 and PQE-9,available from Qiagen; pBS vectors, Phagescript vectors, Bluescriptvectors, pNH8A, pNH16A, pNH18A, pNH46A, available from Stratagene; andptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia.Among preferred eukaryotic vectors are PWLNEO, pSV2CAT, pOG44, pZT1 andpSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL availablefrom Pharmacia. Other suitable vectors will be readily apparent to theskilled artisan.

Among known bacterial promoters for use in the present invention includeE. coli lacI and lacZ promoters, the T3 and T7 promoters, the gptpromoter, the lambda PR, PL promoters and the trp promoter, the HSVthymidine kinase promoter, the early and late SV40 promoters, thepromoters of retroviral LTRs, such as those of the Rous sarcoma virus(“RSV”), and metallothionein promoters, such as the mousemetallothionein-1 promoter.

Transcription of the DNA encoding the polypeptides of the presentinvention by higher eukaryotes may be increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp that act to increase transcriptionalactivity of a promoter in a given host cell-type. Examples of enhancersinclude the SV40 enhancer, which is located on the late side of thereplication origin at bp 100 to 270, the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers.

For secretion of the translated protein into the lumen of theendoplasmic reticulum, into the periplasmic space or into theextracellular environment, appropriate secretation signal may beincorporated into the expressed polypeptide. The signals may beendogenous to the polypeptide or they may be heterologous signals.

The polypeptide may be expressed in a modified form, such as a fusionprotein, and may include not only secretion signals but also additionalheterologous functional regions. Thus, for instance, a region ofadditional amino acids, particularly charged amino acids, may be addedto the N-terminus of the polypeptide to improve stability andpersistence in the host cell, during purification or during subsequenthandling and storage. Also, peptide moieties may be added to thepolypeptide to facilitate purification.

Polypeptides According to the Invention

The invention provides an isolated polypeptide having an amino acidsequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO: 18, anamino acid sequence obtainable by expressing the polynucleotide of SEQID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ IDNO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ IDNO: 11 or SEQ ID NO: 12 in an appropriate host. Also, a peptide orpolypeptide comprising a functional equivalent of the above polypeptidesis comprised within the present invention. The above polypeptides arecollectively comprised in the term “polypeptides according to theinvention”

The terms “peptide” and “oligopeptide” are considered synonymous (as iscommonly recognized) and each term can be used interchangeably as thecontext requires to indicate a chain of at least two amino acids coupledby peptidyl linkages. The word “polypeptide” is used herein for chainscontaining more than seven amino acid residues. All oligopeptide andpolypeptide formulas or sequences herein are written from left to rightand in the direction from amino terminus to carboxy terminus. Theone-letter code of amino acids used herein is commonly known in the artand can be found in Sambrook, et al. (Molecular Cloning: A LaboratoryManual, 2^(nd) , ed. Cold Spring Harbor Laboratory, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989)

By “isolated” polypeptide or protein is intended a polypeptide orprotein removed from its native environment. For example, recombinantlyproduced polypeptides and proteins expressed in host cells areconsidered isolated for the purpose of the invention as are native orrecombinant polypeptides which have been substantially purified by anysuitable technique such as, for example, the single-step purificationmethod disclosed in Smith and Johnson, Gene 67:31-40 (1988).

The amylase according to the invention can be recovered and purifiedfrom recombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. Most preferably, highperformance liquid chromatography “HPLC”) is employed for purification.

Polypeptides of the present invention include naturally purifiedproducts, products of chemical synthetic procedures, and productsproduced by recombinant techniques from a prokaryotic or eukaryotic hostincluding, for example, bacterial, yeast, higher plant, insect andmammalian cells. Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated or may be non-glycosylated. In addition, polypeptides ofthe invention may also include an initial modified methionine residue,in some cases as a result of host-mediated processes.

Protein Fragments

The invention also features biologically active fragments of thepolypeptides according to the invention.

Biologically active fragments of a polypeptide of the invention includepolypeptides comprising amino acid sequences sufficiently identical toor derived from the amino acid sequence of the protein (e.g., the aminoacid sequence of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:16, SEQ ID NO: 17 or SEQ ID NO: 18), which include fewer amino acidsthan the full length protein, and exhibit at least one biologicalactivity of the corresponding full-length protein. Typically,biologically active fragments comprise a domain or motif with at leastone activity of the alpha-amylase. A biologically active fragment of aprotein of the invention can be a polypeptide which is, for example, 10,25, 50, 100 or more amino acids in length. Moreover, other biologicallyactive portions, in which other regions of the protein are deleted, canbe prepared by recombinant techniques and evaluated for one or more ofthe biological activities of the native form of a polypeptide of theinvention.

The invention also features nucleic acid fragments which encode theabove biologically active fragments of the alpha amylase.

Fusion Proteins

The proteins of the present invention or functional equivalents thereof,e.g., biologically active portions thereof, can be operatively linked toa non-alpha-amylase polypeptide (e.g., heterologous amino acidsequences) to form fusion proteins. As used herein, a “chimeric protein”or “fusion protein” comprises an alpha amylase polypeptide operativelylinked to a non-alpha-amylase polypeptide. In a preferred embodiment, afusion protein comprises at least one biologically active fragment of aprotein according to the invention. In this context, the term“operatively linked” is intended to indicate that the alpha amylase andthe non-alpha amylase are fused in-frame to each other either to theN-terminus or C-terminus of the alpha amylase.

For example, in one embodiment, the fusion protein is a GST-fusionprotein in which the alpha amylase sequences are fused to the C-terminusof the GST sequences. Such fusion proteins can facilitate thepurification of recombinant PROTEIN ACCORDING TO THE INVENTION. Inanother embodiment, the fusion protein comprises a protein according tothe invention fused to a heterologous signal sequence at its N-terminus.In certain host cells (e.g., mammalian and Yeast host cells), expressionand/or secretion of a protein according to the invention can beincreased through use of a hetereologous signal sequence.

In another example, the gp67 secretory sequence of the baculovirusenvelope protein can be used as a heterologous signal sequence (CurrentProtocols in Molecular Biology, Ausubel et al., eds., John Wiley & Sons,1992). Other examples of eukaryotic heterologous signal sequencesinclude the secretory sequences of melittin and human placental alkalinephosphatase (Stratagene; La Jolla, Calif.). In yet another example,useful prokarytic heterologous signal sequences include the phoAsecretory signal (Sambrook et al., supra) and the protein A secretorysignal (Pharmacia Biotech; Piscataway, N.J.).

A signal sequence can be used to facilitate secretion and isolation of aprotein or polypeptide of the invention. Signal sequences are typicallycharacterized by a core of hydrophobic amino acids which are generallycleaved from the mature protein during secretion in one or more cleavageevents. Such signal peptides contain processing sites that allowcleavage of the signal sequence from the mature proteins as they passthrough the secretory pathway. The signal sequence directs secretion ofthe protein, such as from a eukaryotic host into which the expressionvector is transformed, and the signal sequence is subsequently orconcurrently cleaved. The protein can then be readily purified from theextracellular medium by art recognized methods. Alternatively, thesignal sequence can be linked to the protein of interest using asequence which facilitates purification, such as with a GST domain.Thus, for instance, the sequence encoding the polypeptide may be fusedto a marker sequence, such as a sequence encoding a peptide, whichfacilitates purification of the fused polypeptide. In certain preferredembodiments of this aspect of the invention, the marker sequence is ahexa-histidine peptide, such as the tag provided in a pQE vector(Qiagen, Inc.), among others, many of which are commercially available.As described in Gentz et al, Proc. Natl. Acad. Sci. USA 86:821-824(1989), for instance, hexa-histidine provides for convenientpurification of the fusion protein. The HA tag is another peptide usefulfor purification which corresponds to an epitope derived of influenzahemaglutinin protein, which has been described by Wilson et al., Cell37:767 (1984), for instance.

Preferably, a chimeric or fusion protein comprising a protein accordingto the invention is produced by standard recombinant DNA techniques. Forexample, DNA fragments coding for the different polypeptide sequencesare ligated together in-frame in accordance with conventionaltechniques, for example by employing blunt-ended or stagger-endedtermini for ligation, restriction enzyme digestion to provide forappropriate termini, filling-in of cohesive ends as appropriate,alkaline phosphatase treatment to avoid undesirable joining, andenzymatic ligation. In another embodiment, the fusion gene can besynthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of gene fragments can becarried out using anchor primers which give rise to complementaryoverhangs between two consecutive gene fragments which can subsequentlybe annealed and reamplified to generate a chimeric gene sequence (see,for example, Current Protocols in Molecular Biology, eds. Ausubel et al.John Wiley & Sons: 1992). Moreover, many expression vectors arecommercially available that already encode a fusion moiety (e.g, a GSTpolypeptide). A nucleic acid according to the invention can be clonedinto such an expression vector such that the fusion moiety is linkedin-frame to the fusion moiety in order to express a fusion proteincomprising a protein according to the invention.

Functional Equivalents

The terms “functional equivalents” and “functional variants” are usedinterchangeably herein. Functional equivalents of the alpha amylaseencoding DNA fragments described herein are isolated DNA fragments thatencode a polypeptide that exhibits a particular function of the A. nigeramylase as defined herein. A functional equivalent of a polypeptideaccording to the invention is a polypeptide that exhibits at least onefunction of an A. niger amylase as defined herein. Functionalequivalents therefore also encompass biologically active fragments.

Functional protein or polypeptide equivalents may contain onlyconservative substitutions of one or more amino acids in the sequencesprovided in SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16,SEQ ID NO: 17 or SEQ ID NO: 18 or substitutions, insertions or deletionsof non-essential amino acids. Accordingly, a non-essential amino acid isa residue that can be altered in SEQ ID NO: 13, SEQ ID NO: 14, SEQ IDNO: 15, SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO: 18 withoutsubstantially altering the biological function. For example, amino acidresidues that are conserved among the proteins of the present invention,are predicted to be particularly unamenable to alteration. Furthermore,amino acids conserved among the proteins according to the presentinvention and other amylases are not likely to be amenable toalteration.

The term “conservative substitution” is intended to mean that asubstitution in which the amino acid residue is replaced with an aminoadd residue having a similar side chain. These families are known in theart and include amino acids with basic side chains (e.g., lysine,arginine and hystidine), acidic side chains (e.g. aspartic acid,glutamic acid), uncharged polar side chains (e.g., glycine, asparagines,glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine tryptophan, histidine).

Functional nucleic acid equivalents may typically contain silentmutations or mutations that do not alter the biological function ofencoded polypeptide. Accordingly, the invention provides nucleic acidmolecules encoding proteins that contain changes in amino acid residuesthat are not essential for a particular biological activity. Suchproteins differ in amino acid sequence from SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO: 18 yetretain at least one biological activity. In one embodiment the isolatednucleic acid molecule comprises a nucleotide sequence encoding aprotein, wherein the protein comprises a substantially homologous aminoacid sequence of at least about 40%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, 99% or more homologous to an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO:15, SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO: 18.

For example, guidance concerning how to make phenotypically silent aminoacid substitutions is provided in Bowie, J. U. et al., Science247:1306-1310 (1990) wherein the authors indicate that there are twomain approaches for studying the tolerance of an amino acid sequence tochange. The first method relies on the process of evolution, in whichmutations are either accepted or rejected by natural selection. Thesecond approach uses genetic engineering to introduce amino acid changesat specific positions of a cloned gene and selects or screens toidentify sequences that maintain functionality. As the authors state,these studies have revealed that proteins are surprisingly tolerant ofamino acid substitutions. The authors further indicate which changes arelikely to be permissive at a certain position of the protein. Forexample, most buried amino acid residues require non-polar side chains,whereas few features of surface side chains are generally conserved.Other such phenotypically silent substitutions are described in Bowie etal, supra, and the references cited therein.

An isolated nucleic acid molecule encoding a protein homologous to theprotein having an amino acid sequence selected from the group consistingof SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ IDNO: 17 or SEQ ID NO: 18 can be created by introducing one or morenucleotide substitutions, additions or deletions into the codingnucleotide sequences having a nucleotide sequence selected from thegroup consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO: 5 or SEQ ID NO: 6 or from the group consisting of SEQ IDNO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQID NO: 12 such that one or more amino acid substitutions, deletions orinsertions are introduced into the encoded protein. Such mutations maybe introduced by standard techniques, such as site-directed mutagenesisand PCR-mediated mutagenesis.

The term “functional equivalents” also encompasses orthologues of the A.niger alpha amylases provided herein. Orthologues of the A. niger alphaamylase are proteins that can be isolated from other strains or speciesand possess a similar or identical biological activity. Such orthologuescan readily be identified as comprising an amino acid sequence that issubstantially homologous to an amino acid sequence selected from thegroup consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ IDNO: 16, SEQ ID NO: 17 or SEQ ID NO: 18.

As defined herein, the term “substantially homologous” refers to a firstamino acid or nucleotide sequence which contains a sufficient or minimumnumber of identical or equivalent (e.g., with similar side chain) aminoacids or nucleotides to a second amino acid or nucleotide sequence suchthat the first and the second amino acid or nucleotide sequences have acommon domain. For example, amino acid or nucleotide sequences whichcontain a common domain having about 40%, preferably 65%, morepreferably 70%, even more preferably 75%, 80%, 85%, 90%, 95%, 96%, 97%,98% or 99% identity or more are defined herein as sufficientlyidentical.

Also, nucleic acids encoding other alpha amylase family members, whichthus have a nucleotide sequence that differs from a sequence selectedfrom the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3,SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 or from the group consistingof SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO:11 or SEQ ID NO: 12, are within the scope of the invention. Moreover,nucleic acids encoding alpha amylases from different species which thushave a nucleotide sequence which differs from a sequence selected fromthe group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ IDNO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 or from the group consisting of SEQID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 orSEQ ID NO: 12 are within the scope of the invention.

Nucleic acid molecules corresponding to variants (e.g. natural allelicvariants) and homologues of the DNA according to the invention can beisolated based on their homology to the nucleic acids disclosed hereinusing the cDNAs disclosed herein or a suitable fragment thereof, as ahybridisation probe according to standard hybridisation techniquespreferably under highly stringent hybridisation conditions.

In addition to naturally occurring allelic variants of the A nigersequences provided herein, the skilled person will recognise thatchanges can be introduced by mutation into the nucleotide sequenceselected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ IDNO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 or from the groupconsisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10,SEQ ID NO: 11 or SEQ ID NO: 12 thereby leading to changes in the aminoacid sequence of the alpha amylase protein without substantiallyaltering the function of the protein.

In another aspect of the invention, improved alpha amylases areprovided. Improved alpha amylases are proteins wherein at least onebiological activity is improved. Such proteins may be obtained byrandomly introducing mutations along all or part of the coding sequence,such as by saturation mutagenesis, and the resulting mutants can beexpressed recombinantly and screened for biological activity. Forinstance, the art provides for standard assays for measuring theenzymatic activity of amylases and thus improved proteins may easily beselected.

In a preferred embodiment the alpha amylase has an amino acid sequencehaving an amino acid sequence selected from the group consisting of SEQID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 prSEQ ID NO: 18. In another embodiment, the alpha amylase is substantiallyhomologous to an amino acid sequence selected from the group consistingof SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ IDNO: 17 or SEQ ID NO: 18 and retains at least one biological activity ofa polypeptide having an amino acid sequence selected from the groupconsisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:16, SEQ ID NO: 17 or SEQ ID NO: 18, yet differs in amino acid sequencedue to natural variation or mutagenesis as described above.

In a further preferred embodiment, the alpha amylase has an amino acidsequence encoded by an isolated nucleic acid fragment capable ofhybridising to a nucleic acid having a nucleotide sequence selected fromthe group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ IDNO: 4, SEQ ID NO:5 or SEQ ID NO: 6 or from the group consisting of SEQID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 orSEQ ID NO: 12, preferably under highly stringent hybridisationconditions.

Accordingly, an alpha amylase according to the invention is an isolatedprotein which comprises an amino acid sequence at least about 40%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homologous toan amino acid sequence selected from the group consisting of SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 or SEQ IDNO: 18 and retains at least one functional activity of the polypeptidehaving an amino acid sequence selected from the group consisting of SEQID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO:17 orSEQ ID NO: 18.

Functional equivalents of a protein according to the invention can alsobe identified e.g. by screening combinatorial libraries of mutants, e.g.truncation mutants, of the protein of the invention for amylaseactivity. In one embodiment, a variegated library of variants isgenerated by combinatorial mutagenesis at the nucleic acid level. Avariegated library of variants can be produced by, for example,enzymatically ligating a mixture of synthetic oligonucleotides into genesequences such that a degenerate set of potential protein sequences isexpressible as individual polypeptides, or alternatively, as a set oflarger fusion proteins (e.g., for phage display). There are a variety ofmethods that can be used to produce libraries of potential variants ofthe polypeptides of the invention from a degenerate oligonucleotidesequence. Methods for synthesizing degenerate oligonucleotides are knownin the art (see, e.g., Narang (1983) Tetrahedron 39:3; Itakura et al.(1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science198:1056; Ike et al. (1983) Nucleic Acid Res. 11:477).

In addition, libraries of fragments of the coding sequence of apolypeptide of the invention can be used to generate a variegatedpopulation of polypeptides for screening a subsequent selection ofvariants. For example, a library of coding sequence fragments can begenerated by treating a double stranded PCR fragment of the codingsequence of interest with a nuclease under conditions wherein nickingoccurs only about once per molecule, denaturing the double stranded DNA,renaturing the DNA to form double stranded DNA which can includesense/antisense pairs from different nicked products, removing singlestranded portions from reformed duplexes by treatment with S1 nuclease,and ligating the resulting fragment library into an expression vector.By this method, an expression library can be derived which encodesN-terminal and internal fragments of various sizes of the protein ofinterest.

Several techniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations of truncation, and forscreening cDNA libraries for gene products having a selected property.The most widely used techniques, which are amenable to high through-putanalysis, for screening large gene libraries typically include cloningthe gene library into replicable expression vectors, transformingappropriate cells with the resulting library of vectors, and expressingthe combinatorial genes under conditions in which detection of a desiredactivity facilitates isolation of the vector encoding the gene whoseproduct was detected. Recursive ensemble mutagenesis (REM), a techniquewhich enhances the frequency of functional mutants in the libraries, canbe used in combination with the screening assays to identify variants ofa protein of the invention (Arkin and Yourvan (1992) Proc. Natl. Acad.Sci. USA 89:7811-7815; Delgrave et al. (1993) Protein Engineering6(3):327-331).

It will be apparent for the person skilled in the art that DNA sequencepolymorphisms may exist that may lead to changes in the amino acidsequence of the alpha amylase within a given population. Such geneticpolymorphisms may exist in cells from different populations or within apopulation due to natural allelic variation. Allelic variants may alsoinclude functional equivalents.

Fragments of a polynucleotide according to the invention may alsocomprise polynucleotides not encoding functional polypeptides. Suchpolynucleotides may function as probes or primers for a PCR reaction.

Nucleic acids according to the invention irrespective of whether theyencode functional or non-functional polypeptides, can be used ashybridization probes or polymerase chain reaction (PCR) primers. Uses ofthe nucleic acid molecules of the present invention that do not encode apolypeptide having alpha amylase activity include, inter alia, (1)isolating the gene encoding the alpha amylase, or allelic variantsthereof from a cDNA library e.g. from other organisms than A niger; (2)in situ hybridization (e.g. FISH) to metaphase chromosomal spreads toprovide precise chromosomal location of the alpha amylase gene asdescribed in Verma et al., Human Chromosomes: a Manual of BasicTechniques, Pergamon Press, New York (1988); (3) Northern blot analysisfor detecting expression of alpha amylase mRNA in specific tissuesand/or cells and 4) probes and primers that can be used as a diagnostictool to analyse the presence of a nucleic acid hybridisable to the alphaamylase probe in a given biological (e.g. tissue) sample.

Also encompassed by the invention is a method of obtaining a functionalequivalent of an alpha amylase gene or cDNA. Such a method entailsobtaining a labelled probe that includes an isolated nucleic acid whichencodes all or a portion of the sequence having an amino acid sequenceselected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO: 18 or a variantthereof; screening a nucleic acid fragment library with the labelledprobe under conditions that allow hybridisation of the probe to nucleicacid fragments in the library, thereby forming nucleic acid duplexes,and preparing a full-length gene sequence from the nucleic acidfragments in any labelled duplex to obtain a gene related to the alphaamylase gene.

In one embodiment, a nucleic acid according to the invention is at least40%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or more homologous to a nucleic acid sequence shown in SEQ IDNO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 SEQ ID NO:6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO:11 or SEQ ID NO: 12 or the complement thereof.

In another preferred embodiment a polypeptide of the invention is atleast 40%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or more homologous to the amino acid sequence shown inSEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO:17 or SEQ ID NO: 18.

Host Cells

In another embodiment, the invention features cells, e.g., transformedhost cells or recombinant host cells that contain a nucleic acidencompassed by the invention. A “transformed cell” or “recombinant cell”is a cell into which (or into an ancestor of which) has been introduced,by means of recombinant DNA techniques, a nucleic acid according to theinvention. Both prokaryotic and eukaryotic cells are included, e.g.,bacteria, fungi, yeast, and the like, especially preferred are cellsfrom filamentous fungi, in particular Aspergillus niger.

A host cell can be chosen that modulates the expression of the insertedsequences, or modifies and processes the gene product in a specific,desired fashion. Such modifications (e.g., glycosylation) and processing(e.g., cleavage) of protein products may facilitate optimal functioningof the protein.

Various host cells have characteristic and specific mechanisms forpost-translational processing and modification of proteins and geneproducts. Appropriate cell lines or host systems familiar to those ofskill in the art of molecular biology and/or microbiology can be chosento ensure the desired and correct modification and processing of theforeign protein expressed. To this end, eukaryotic host cells thatpossess the cellular machinery for proper processing of the primarytranscript, glycosylation, and phosphorylation of the gene product canbe used. Such host cells are well known in the art.

Host cells also include, but are not limited to, mammalian cell linessuch as CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, W138, and choroidplexus cell lines.

If desired, the polypeptides according to the invention can be producedby a stably-transfected cell line. A number of vectors suitable forstable transfection of mammalian cells are available to the public,methods for constructing such cell lines are also publicly known, e.g.,in Ausubel et al. (supra).

Antibodies

The invention further features antibodies, such as monoclonal orpolyclonal antibodies, that specifically bind alpha amylases accordingto the invention.

As used herein, the term “antibody” (Ab) or “monoclonal antibody” (Mab)is meant to include intact molecules as well as antibody fragments (suchas, for example, Fab and F(ab′)₂ fragments) which are capable ofspecifically binding to a protein according to the invention. Fab andF(ab′)₂ fragments lack the Fc fragment of intact antibody, dear morerapidly from the circulation, and may have less non-specific tissuebinding of an intact antibody (Wahl et al., J. Nucl. Med. 24:316-325(1983)). Thus, these fragments are preferred.

The antibodies of the present invention may be prepared by any of avariety of methods. For example, cells expressing the alpha amylaseaccording to the invention or an antigenic fragment thereof can beadministered to an animal in order to induce the production of seracontaining polyclonal antibodies. In a preferred method, a preparationof a protein according to the invention is prepared and purified torender it substantially free of natural contaminants. Such a preparationis then introduced into an animal in order to produce polyclonalantisera of greater specific activity. In the most preferred method, theantibodies of the present invention are monoclonal antibodies (or alphaamylase-binding fragments thereof). Such monoclonal antibodies can beprepared using hybridoma technology (Kohler et al., Nature 256:495(1975); Kohler et al., Eur. J. Immunol. 6:511 (1976); Hammerling et al.,In: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y., pp.563-681 (1981)). In general, such procedures involve immunizing ananimal (preferably a mouse) with a protein according to the inventionor, with a cell expressing a protein according to the invention. Thesplenocytes of thus immunised mice are extracted and fused with asuitable myeloma cell line. Any suitable myeloma cell line may beemployed in accordance with the present inventoin; however, it ispreferably to employ the parent myeloma cell line (SP₂O), available fromthe American Type Culture Collection, Rockville, Md. After fusion, theresulting hybridoma cells are selectively maintained in HAT medium, andthen cloned by limiting dilution as described by Wands et al.(Gastro-enterology 80:225-232 (1981)). The hybridoma cells obtainedthrough such a selection are then assayed to identify clones whichsecrete antibodies capable of binding the alpha amylase antigen. Ingeneral, the polypeptides can be coupled to a carrier protein, such asKLH, as described in Ausubel et al., supra, mixed with an adjuvant, andinjected into a host mammal.

In particular, various host animals can be immunized by injection of apolypeptide of interest Examples of suitable host animals includerabbits, mice, guinea pigs, and rats. Various adjuvants can be used toincrease the immunological response, depending on the host species,including but not limited to Freund's (complete and incomplete),adjuvant mineral gels such as aluminum hydroxide, surface actvesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, keyhole limpet hemocyanin, dinitrophenol, BCG (bacilleCalmette-Guerin) and Corynebacterium parvum. Polyclonal antibodies areheterogeneous populations of antibody molecules derived from the sera ofthe immunized animals.

Such antibodies can be of any immunoglobulin class including IgG, IgM,IgE, IgA, IgD, and any subclass thereof. The hybridomas producing themAbs of this invention can be cultivated in vitro or in vivo.

Once produced, polyclonal or monoclonal antibodies are tested forspecific recognition of a protein according to the invention orfunctional equivalent thereof in an immunoassay, such as a Western blotor immunoprecipitation analysis using standard techniques, e.g., asdescribed in Ausubel et al., supra. Antibodies that specifically bind toa protein according to the invention or functional equivalents thereofare useful in the invention. For example, such antibodies can be used inan immunoassay to detect a protein according to the invention inpathogenic or non-pathogenic strains of Aspergillus (e.g., inAspergillus extracts).

Preferably, antibodies of the invention are produced using fragments ofa protein according to the invention that appears likely to beantigenic, by criteria such as high frequency of charged residues. Forexample, such fragments may be generated by standard techniques of PCR,and then cloned into the pGEX expression vector (Ausubel et al., supra).Fusion proteins may then be expressed in E. coli and purified using aglutathione agarose affinity matrix as described in Ausubel, et al.,supra. If desired, several (e.g., two or three) fusions can be generatedfor each protein, and each fusion can be injected into at least tworabbits. Antisera can be raised by injections in a series, typicallyincluding at least three booster injections. Typically, the antisera arechecked for their ability to immunoprecipitate a recombinant alphaamylase according to the invention or functional equivalents thereofwhereas unrelated proteins may serve as a control for the specificity ofthe immune reaction.

Alternatively, techniques decribed for the production of single chainantibodies (U.S. Pat. Nos. 4,946,778 and 4,704,692) can be adapted toproduce single chain antibodies against a protein according to theinvention or functional equivalents thereof. Kits for generating andscreening phage display libraries are commercially available, e.g. fromPharmacia.

Additionally, examples of methods and reagents particularly amenable foruse in generating and screening antibody display library can be foundin, for example, U.S. Pat. No. 5,223,409; PCT Publication No. WO92/18619; PCT Publication No. WO 91/17271; PCT Publication No. WO 20791;PCT Publication No. WO 92/20791; PCT Publication No. WO 92/15679; PCTPublication No. WO 93/01288; PCT Publication No. WO 92/01047; PCTPublication No. WO 92/09690; PCT Publication No. WO 90/02809; Fuchs etal. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum. Antibod.Hybridomas 3:81-85; Huse et al. (1989) Science 246;1275-1281; Griffithset al. (1993) EMBO J. 12:725-734.

Polyclonal and monoclonal antibodies that specifically bind a proteinaccording to the invention or functional equivalents thereof can beused, for example, to detect expression of gene encoding a proteinaccording to the invention or a functional equivalent thereof e.g. inanother strain of Aspergillus. For example, a protein according to theinvention can be readily detected in conventional immunoassays ofAspergillus cells or extracts. Examples of suitable assays include,without limitation, Western blotting, ELISAs, radioimmune assays, andthe like.

By “specifically binds is meant that an antibody recognizes and binds aparticular antigen, e.g., a protein according to the inventionpolypeptide, but does not substantially recognize and bind otherunrelated molecules in a sample.

Antibodies can be purified, for example, by affinity chromatographymethods in which the polypeptide antigen is immobilized on a resin.

An antibody directed against a polypeptide of the invention (e.g.,monoclonal antibody) can be used to isolate the polypeptide by standardtechniques, such as affinity chromatography or immunoprecipitation.Moreover, such an antibody can be used to detect the protein (e.g., in acellular lysate or cell supernatant) in order to evaluate the abundanceand pattern of expression of the polypeptide. The antibodies can also beused diagnostically to monitor protein levels in cells or tissue as partof a clinical testing procedure, e.g., to, for example, determine theefficacy of a given treatment regimen or in the diagnosis ofAspergillosis.

Detection can be facilitated by coupling the antibody to a detectablesubstance. Examples of detectable substances include various enzymes,prosthetic groups, fluorescent materials, luminescent materials,bioluminescent materials, and radioactive materials. Examples ofsuitable enzymes include horseradish peroxidase, alkaline phosphatase,β-galactosidase, or acetylcholinesterase; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin, and examples of suitable radioactive materialsinclude ¹²⁵I, ¹³¹I, ³⁵S or ³H.

Preferred epitopes encompassed by the antigenic peptide are regions thatare located on the surface of the protein, e.g. hydrophilic regions.Hydrophobicity plots of the proteins of the invention can be used toidentify hydrophilic regions.

The antigenic peptide of a protein of the invention comprises at least 7(preferably 10, 15, 20, or 30) contiguous amino acid residues of anamino acid sequense selected from the group consisting of SEQ ID NO: 13,SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO:18 and encompasses an epitope of the protein such that an antibodyraised against the peptide forms a specific immune complex with theprotein.

Preferred epitopes encompassed by the antigenic peptide are regions of aprotein according to the invention that are located on the surface ofthe protein, e.g., hydrophilic regions, hydrophobic regions, alpharegions, beta regions, coil regions, turn regions and flexible regions.

Immunoassays

Qualitative or quantitative determination of a polypeptide according tothe present invention in a biological sample can occur using anyart-known method. Antibody-based techniques provide special advantagesfor assaying specific polypeptide levels in a biological sample.

In these, the specific recognition is provided by the primary antibody(polyclonal or monoclonal) but the secondary detection system canutilize fluorescent, enzyme, or other conjugated secondary antibodies.As a result, an immunocomplex is obtained.

Accordingly, the invention provides a method for diagnosing whether acertain organism is infected with Aspergillus comprising the steps of:

-   -   Isolating a biological sample from said organism suspected to be        infected with Aspergillus,    -   reacting said biological sample with an antibody according to        the invention,    -   determining whether immune complexes are formed.

Tissues can also be extracted, e.g., with urea and neutral detergent,for the liberation of protein for Western-blot or dot/slot assay. Thistechnique can also be applied to body fluids.

Other antibody-based methods useful for detecting a protein according tothe invention, include immunoassays, such as the enzyme linkedimmunosorbent assay (ELISA) and the radioimmunoassay (RIA). For example,specific monoclonal antibodies against a protein according to theinvention can be used both as an immunoabsorbent and as anenzyme-labeled probe to detect and quantify a protein according to theinvention. The amount of specific protein present in the sample can becalculated by reference to the amount present in a standard preparationusing a linear regression computer algorithm. In another ELISA assay,two distinct specific monoclonal antibodies can be used to detect aprotein according to the invention in a biological fluid. In this assay,one of the antibodies is used as the immuno-absorbent and the other asthe enzyme-labeled probe.

The above techniques may be conducted essentially as a “one-step” or“two-step” assay. The “one-step” assay involves contacting a proteinaccording to the invention with immobilized antibody and, withoutwashing, contacting the mixture with the labeled antibody. The“two-step” assay involves washing before contacting the mixture with thelabeled antibody. Other conventional methods may also be employed assuitable. It is usually desirable to immobilize one component of theassay system on a support, thereby allowing other components of thesystem to be brought into contact with the component and readily removedfrom the sample.

Suitable enzyme labels include, for example, those from the oxidasegroup, which catalyze the production of hydrogen peroxide by reactingwith substrate. Activity of an oxidase label may be assayed by measuringthe concentration of hydrogen peroxide formed by the enzyme-labelledantibody/substrate reaction.

Besides enzymes, other suitable labels include radioisotopes, such asiodine (¹²¹I, ¹²¹I), carbon (¹⁴C), sulphur (³⁵S), tritium (³H), indium(¹¹²In), and technetium (^(99m)Tc), and fluorescent labels, such asfluorescein and rhodamine, and biotin.

Specific binding of a test compound to a protein according to theinvention can be detected, for example, in vitro by reversibly orirreversibly immobilizing a protein according to the inventionpolypeptide on a substrate, e.g., the surface of a well of a 96-wellpolystyrene microtitre plate. Methods for immobilizing polypeptides andother small molecules are well known in the art. For example, themicrotitre plates can be coated with a protein according to theinvention by adding the polypeptide in a solution (typically, at aconcentration of 0.05 to 1 mg/ml in a volume of 1-100 ul) to each well,and incubating the plates at room temperature to 37° C. for 0.1 to 36hours. Polypeptides that are not bound to the plate can be removed byshaking the excess solution from the plate, and then washing the plate(once or repeatedly) with water or a buffer. Typically, the polypeptideis contained in water or a buffer. The plate is then washed with abuffer that lacks the bound polypeptide. To block the freeprotein-binding sites on the plates, the plates are blocked with aprotein that is unrelated to the bound polypeptide. For example, 300 ulof bovine serum albumin (BSA) at a concentration of 2 mg/ml in Tris-HClis suitable. Suitable substrates include those substrates that contain adefined cross-linking chemistry (e.g., plastic substrates, such aspolystyrene, styrene, or polypropylene substrates from Corning CostarCorp. (Cambridge, Mass.), for example). If desired, a beaded particle,e.g., beaded agarose or beaded sepharose, can be used as the substrate.

Binding of the test compound to the polypeptides according to theinvention can be detected by any of a variety of art known methods. Forexample, a specific antibody can be used in an immunoassay. If desired,the antibody can be labeled (e.g., fluorescently or with a radioisotope)and detected directly (see, e.g., West and McMahon, J. Cell Biol.74:264, 1977). Alternatively, a second antibody can be used fordetection (e.g., a labeled antibody that binds the Fc portion of ananti-AN97 antibody). In an alternative detection method, a proteinaccording to the invention is labelled (e.g., with a radioisotope,fluorophore, chromophore, or the like), and the label is detected. Instill another method, a protein according to the invention is producedas a fusion protein with a protein that can be detected optically, e.g.,green fluorescent protein (which can be detected under UV light). In analternative method, a protein according to the invention polypeptide canbe covalently attached to or fused with an enzyme having a detectableenzymatic activity, such as horse radish peroxidase, alkalinephosphatase, a-galactosidase, or glucose oxidase. Genes encoding all ofthese enzymes have been cloned and are readily available for use bythose of skill in the art. If desired, the fusion protein can include anantigen, and such an antigen can be detected and measured with apolyclonal or monoclonal antibody using conventional methods. Suitableantigens include enzymes (e.g., horse radish peroxidase, alkalinephosphatase, and a-galactosidase) and non-enzymatic polypeptides (e.g.,serum proteins, such as BSA and globulins, and milk proteins, such ascaseins).

Epitopes, Antigens and Immunogens.

In another aspect, the invention provides a peptide or polypeptidecomprising an epitope-bearing portion of a polypeptide of the invention.The epitope of this polypeptide portion is an immunogenic or antigenicepitope of a polypeptide of the invention. An immunogenic epitope” isdefined as a part of a protein that elicits an antibody response whenthe whole protein is the immunogen. These immunogenic epitopes arebelieved to be confined to a few loci on the molecule. On the otherhand, a region of a protein molecule to which an antibody can bind isdefined as an “antigenic epitope.” The number of immunogenic epitopes ofa protein generally is less than the number of antigenic epitopes. See,for instance, Geysen, H. M. et al., Proc. Natl. Acad. Sci. USA81:3998-4002 (1984).

As to the selection of peptides or polypeptides bearing an antigenicepitope (i.e., that contain a region of a protein molecule to which anantibody can bind), it is well known in that art that relatively shortsynthetic peptides that mimic part of a protein sequence are routinelycapable of eliciting an antiserum that reacts with the partiallymimicked protein. See, for instance, Sutcliffe, J. G. et al., Science219:660-666 (1984). Peptides capable of eliciting protein-reactive seraare frequently represented in the primary sequence of a protein, can becharacterized by a set of simple chemical rules, and are confinedneither to immunodominant regions of intact proteins (i.e., immunogenicepitopes) nor to the amino or carboxyl terminals. Peptides that areextremely hydrophobic and those of six or fewer residues generally areineffective at inducing antibodies that bind to the mimicked protein;longer, soluble peptides, especially those containing proline residues,usually are effective. Sutcliffe et al., supra, at 661. For instance, 18of 20 peptides designed according to these guidelines, containing 8-39residues covering 75% of the sequence of the influenza virushemagglutinin HAI polypeptide chain, induced antibodies that reactedwith the HA1 protein or intact virus; and 12/12 peptides from the MuLVpolymerase and 18/18 from the rabies glycoprotein induced antibodiesthat precipitated the respective proteins.

Antigenic epitope-bearing peptides and polypeptides of the invention aretherefore useful to raise antibodies, including monoclonal antibodies,that bind specifically to a polypeptide of the invention. Thus, a highproportion of hybridomas obtained by fusion of spleen cells from donorsimmunized with an antigen epitope-bearing peptide generally secreteantibody reactive with the native protein. Sutcliffe et al., supra, at663. The antibodies raised by antigenic epitope bearing peptides orpolypeptides are useful to detect the mimicked protein, and antibodiesto different peptides may be used for tracking the fate of variousregions of a protein precursor which undergoes posttranslationprocessing. The peptides and antipeptide antibodies may be used in avariety of qualitative or quantitative assays for the mimicked protein,for instance in competition assays since it has been shown that evenshort peptides (e.g., about 9 amino acids) can bind and displace thelarger peptides in immunoprecipitation assays. See, for instance,Wilson, I. A. et al., Cell 37:767-778 at 777 (1984). The antipeptideantibodies of the invention also are useful for purification of themimicked protein, for instance, by adsorption chromatography usingmethods well known in the art.

Antigenic epitope-bearing peptides and polypeptides of the inventiondesigned according to the above guidelines preferably contain a sequenceof at least seven, more preferably at least nine and most preferablybetween about 15 to about 30 amino acids contained within the amino acidsequence of a polypeptide of the invention. However, peptides orpolypeptides comprising a larger portion of an amino acid sequence of apolypeptide of the invention, containing about 30 to about 50 aminoacids, or any length up to and including the entire amino acid sequenceof a polypeptide of the invention, also are considered epitope-bearingpeptides or polypeptides of the invention and also are useful forinducing antibodies that react with the mimicked protein. Preferably,the amino acid sequence of the epitope-bearing peptide is selected toprovide substantial solubility in aqueous solvents (i.e., the sequenceincludes relatively hydrophilic residues and highly hydrophobicsequences are preferably avoided); and sequences containing prolineresidues are particularly preferred.

The epitope-bearing peptides and polypeptides of the invention may beproduced by any conventional means for making peptides or polypeptidesincluding recombinant means using nucleic acid molecules of theinvention. For instance, a short epitope-bearing amino acid sequence maybe fused to a larger polypeptide which acts as a carrier duringrecombinant production and purification, as well as during immunizationto produce anti-peptide antibodies.

Epitope-bearing peptides also may be synthesized using known methods ofchemical synthesis. For instance, Houghten has described a simple methodfor synthesis of large numbers of peptides, such as 10-20 mg of 248different 13 residue peptides representing single amino acid variants ofa segment of the HAI polypeptide which were prepared and characterized(by ELISA-type binding studies) in less than four weeks. Houghten, R.A., Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985). This “SimultaneousMultiple Peptide Synthesis (SMPS)” process is further described in U.S.Pat. No. 4,631,211 to Houghten et al. (1986). In this procedure theindividual resins for the solid-phase synthesis of various peptides arecontained in separate solvent-permeable packets, enabling the optimaluse of the many identical repetitive steps involved in solid-phasemethods.

A completely manual procedure allows 500-1000 or more syntheses to beconducted simultaneously. Houghten et al., supra, at 5134.

Epitope-bearing peptides and polypeptides of the invention are used toinduce antibodies according to methods well known in the art. See, forinstance, Sutcliffe et al., supra; Wilson et al., supra; Chow, M. etal., Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle, F. J. et al., J.Gen. Virol. 66:2347-2354 (1985).

Generally, animals may be immunized with free peptide; however,anti-peptide antibody titer may be boosted by coupling of the peptide toa macromolecular carrier, such as keyhole limpet hemocyanin (KLH) ortetanus toxoid. For instance, peptides containing cysteine may becoupled to carrier using a linker such asmaleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptidesmay be coupled to carrier using a more general linking agent such asglutaraldehyde.

Animals such as rabbits, rats and mice are immunized with either free orcarrier coupled peptides, for instance, by intraperitoneal and/orintradermal injection of emulsions containing about 100 ug peptide orcarrier protein and Freund's adjuvant. Several booster injections may beneeded, for instance, at intervals of about two weeks, to provide auseful titer of anti-peptide antibody which can be detected, forexample, by ELISA assay using free peptide adsorbed to a solid surface.The titer of anti-peptide antibodies in serum from an immunized animalmay be increased by selection of anti-peptide antibodies, for instance,by adsorption to the peptide on a solid support and elution of theselected antibodies according to methods well known in the art.

Immunogenic epitope-bearing peptides of the invention, i.e., those partsof a protein that elicit an antibody response when the whole protein isthe immunogen, are identified according to methods known in the art. Forinstance, Geysen et al., 1984, supra, discloses a procedure for rapidconcurrent synthesis on solid supports of hundreds of peptides ofsufficient purity to react in an enzyme-linked immunosorbent assay.Interaction of synthesized peptides with antibodies is then easilydetected without removing them from the support. In this manner apeptide bearing an immunogenic epitope of a desired protein may beidentified routinely by one of ordinary skill in the art. For instance,the immunologically important epitope in the coat protein offoot-and-mouth disease virus was located by Geysen et al. with aresolution of seven amino acids by synthesis of an overlapping set ofall 208 possible hexapeptides covering the entire 213 amino acidsequence of the protein. Then, a complete replacement set of peptides inwhich all 20 amino acids were substituted in turn at every positionwithin the epitope were synthesized, and the particular amino acidsconferring specificity for the reaction with antibody were determined.Thus, peptide analogs of the epitope-bearing peptides of the inventioncan be made routinely by this method. U.S. Pat. No. 4,708,781 to Geysen(1987) further describes this method of identifying a peptide bearing animmunogenic epitope of a desired protein.

Further still, U.S. Pat. No. 5,194,392 to Geysen (1990) describes ageneral method of detecting or determining the sequence of monomers(amino acids or other compounds) which is a topological equivalent ofthe epitope (i.e., a “mimotope”) which is complementary to a particularparatope (antigen binding site) of an antibody of interest. Moregenerally, U.S. Pat. No. 4,433,092 to Geysen (1989) describes a methodof detecting or determining a sequence of monomers which is atopographical equivalent of a ligand which is complementary to theligand binding site of a particular receptor of interest. Similarly,U.S. Pat. No. 5,480,971 to Houghten, R. A et al. (1996) on PeralkylatedOligopeptide Mixtures discloses linear C₁-C₇-alkyl peralkylatedoligopeptides and sets and libraries of such peptides, as well asmethods for using such oligopeptide sets and libraries for determiningthe sequence of a peralkylated oligopeptide that preferentially binds toan acceptor molecule of interest. Thus, non-peptide analogs of theepitope-bearing peptides of the invention also can be made routinely bythese methods.

Use of Alpha Amylases in Industrial Processes

The invention also relates to the use of a protein according to theinvention in a selected number of industrial and pharmaceuticalprocesses. Despite the long term experience obtained with theseprocesses, the amylase according to the invention features a number ofsignificant advantages over the enzymes currently used. Depending on thespecific application, these advantages can include aspects like lowerproduction costs, higher specificity towards the substrate, lessantigenic, less undesirable side activities, higher yields when producedin a suitable microorganism, more suitable pH and temperature ranges,better tastes of the final product as well as food grade and kosheraspects.

An important aspect of the amylases according to the invention is thatthey cover a whole range of pH and temperature optima which are ideallysuited for a variety of applications. For example many large scaleprocesses benefit from relatively high processing temperatures of 50degrees C. or higher, e.g. to control the risks of microbial infections.Several alpha amylases according to the invention comply with thisdemand but at the same time they are not that heat stable that theyresist attempts to inactivate the enzyme by an additional heattreatment. The latter feature allows production routes that yield finalproducts free of residual enzyme activity. Similarly many feed and foodproducts have slightly acidic pH values so that amylases with acidic ornear neutral pH optima are preferred for their processing. An alphaamylase according to the invention complies with this requirement aswell.

1-22. (canceled)
 23. An isolated polypeptide that has amylase activityand has an amino acid sequence at least 40% homologous to a sequenceselected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO:
 18. 24. Theisolated polypeptide of claim 23 which has an amino acid sequence atleast 70% homologous to a sequence selected from the group consisting ofSEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO:17 and SEQ ID NO:
 18. 25. The isolated polypeptide of claim 23 which hasan amino acid sequence at least 80% homologous to a sequence selectedfrom the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO:15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO:
 18. 26. The isolatedpolypeptide of claim 23 which has an amino acid sequence at least 90%homologous to a sequence selected from the group consisting of SEQ IDNO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 andSEQ ID NO:
 18. 27. The isolated polypeptide of claim 23 which has anamino acid sequence at least 95% homologous to a sequence selected fromthe group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQID NO: 16, SEQ ID NO: 17 and SEQ ID NO:
 18. 28. The polypeptide of claim23 that has the sequence of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15,SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO:
 18. 29. The polypeptide ofclaim 23 obtainable from Aspergillus niger.
 30. An isolatedpolynucleotide encoding at least one functional domain of thepolypeptide of claim
 23. 31. An isolated polynucleotide encoding atleast one functional domain of the polypeptide of claim
 28. 32. Anisolated polynucleotide encoding the polypeptide of claim
 23. 33. Anisolated polynucleotide encoding the polypeptide of claim
 24. 34. Anisolated polynucleotide encoding the polypeptide of claim
 25. 35. Anisolated polynucleotide encoding the polypeptide of claim
 26. 36. Anisolated polynucleotide encoding the polypeptide of claim
 27. 37. Anisolated polynucleotide encoding the polypeptide of claim
 28. 38. Thepolynucleotide of claim 30 hybridizable under high stringency conditionsto a polynucleotide selected from the group consisting of SEQ ID NO: 1,SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 SEQ ID NO: 6 orfrom the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9,SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12 and their complements.39. The polynucleotide of claim 32 hybridizable under high stringencyconditions to a polynucleotide selected from the group consisting of SEQID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQID NO: 6 or from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQID NO: 9, SEQ ID NO: 10, SEQ ID NO: 1 and SEQ ID NO: 12 and theircomplements.
 40. The polynucleotide of claim 39 selected from the groupconsisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,SEQ ID NO: 5 and SEQ ID NO: 6 or from the group consisting of SEQ ID NO:7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ IDNO: 12 and their complements.
 41. The polynucleotide of claim 39obtainable from a filamentous fungus.
 42. The polynucleotide of claim 41obtainable from A. niger.
 43. A vector comprising a polynucleotidesequence of claim
 30. 44. A vector comprising a polynucleotide sequenceof claim
 32. 45. A recombinant nucleic acid molecule wherein thepolynucleotide sequence of claim 30 is operatively linked withregulatory sequences suitable for expression of said polynucleotidesequence in a suitable host cell.
 46. A recombinant nucleic acidmolecule wherein the polynucleotide sequence of claim 32 is operativelylinked with regulatory sequences suitable for expression of saidpolynucleotide sequence in a suitable host cell.
 47. A recombinant hostcell comprising the nucleic acid molecule of claim
 45. 48. A recombinanthost cell comprising the nucleic acid molecule of claim
 46. 49. A methodfor manufacturing an amylase comprising culturing said cell of claim 47under conditions allowing expression of said polynucleotide sequence andoptionally purifying the encoded amylase from said cell or culturemedium.
 50. A method for manufacturing an amylase comprising culturingsaid cell of claim 48 under conditions allowing expression of saidpolynucleotide sequence and optionally purifying the encoded amylasefrom said cell or culture medium.
 51. A recombinant host cell expressinga polypeptide of claim
 23. 52. A recombinant host cell expressing apolypeptide of claim
 28. 53. Purified antibodies reactive with thepolypeptide of claim
 23. 54. Purified antibodies reactive with thepolypeptide of claim
 28. 55. A fusion protein comprising the polypeptideof claim
 23. 56. A fusion protein comprising the polypeptide of claim28.
 57. Recombinant amylase comprising a functional domain of thepolypeptide of claim
 23. 58. Recombinant amylase comprising a functionaldomain of the polypeptide of claim
 28. 59. An isolated polynucleotide,comprising at least 12 consecutive nucleotides of a nucleotide sequencethat hybridizes under stringent conditions to a sequence selected fromthe group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ IDNO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 or from the group consisting of SEQID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 1 andSEQ ID NO: 12, and their complements.
 60. The isolated polynucleotide ofclaim 59 comprising at least 12 consecutive nucleotides of a sequenceselected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ IDNO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 or from the groupconsisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10,SEQ ID NO: 11 and SEQ ID NO: 12, and their complements.
 61. A method formanufacturing a polynucleotide according to claim 30 comprisingculturing a host cell transformed with said polynucleotide and isolatingsaid polynucleotide from said host cell.
 62. A method for manufacturinga polynucleotide according to claim 32 comprising culturing a host celltransformed with said polynucleotide and isolating said polynucleotidefrom said host cell.